The long-term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria, and its relationship to mitochondrial mutagenesis and human disease. A combined approach of current methods in biochemistry and molecular genetics is being pursued to study the mechanism, structure and physiology of the major replicative enzyme in mitochondria, DNA polymerase y (pol y), and its functional interactions with proteins at the mitochondrial replication fork. Structure-function studies of pol y will involve comparative kinetic and physical analyses of native and altered forms, to continue to elucidate the role of the small subunit in enzyme function, and to dissect functional domains in both the catalytic and accessory subunits. Mutational studies will focus on elucidating the roles of the spacer region of the catalytic core, and physical analyses will map subunit contacts. Physiological studies will focus on expanding the development of a transgenic fly model to examine the relationship between pol y function, mitochondrial DNA replication fidelity, aging and disease. A novel recombinational knock-in approach will be taken to introduce and study several human-disease-related alleles of pol y in the context of mitochondrial biogenesis. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging, and disease. Mitochondrial biogenesis proceeds in parallel with cell proliferation, but it is neither tightly coupled to mitochondrial DNA replication nor to the cell cycle. Nevertheless, because both the DNA content of the mitochondrion and the number of mitochondria in cells remain relatively constant, specific regulatory mechanisms are likely required to couple mitochondrial DNA replication and biogenesis to nuclear DNA replication and cell division. A detailed analysis of the key enzyme involved in mitochondrial DNA replication, DNA polymerase y, will represent a major contribution toward an eventual understanding of mitochondrial biogenesis and function in normal and diseased tissues. ! A variety of mitochondrial DNA diseases have recently been documented. This, and an increased recognition that antiviral and anti-tumor drugs frequently affect mitochondrial DNA function, and in particular, the activity of pol y, demonstrate a critical need for an in-depth understanding of this essential cellular DNA polymerase, and other proteins that function at the replication fork.